In vivo stain composition, process of manufacture, and methods of use to identify dysplastic tissue

ABSTRACT

N-demethylated and N,N-demethylated derivatives of toluidine blue O and compositions which include these derivatives and the conformational isomers of toluidine blue O. Improved methods for the detection of dysplastic oral tissue using such compositions. 
     Processes for synthesis of toluidine blue O products, in which a complexing agent is introduced prior to the last stage of oxidation of a three-step synthesis from N,N-dimethyl-ρ-phenylenediamine. 
     An HPLC method for characterizing toluidine blue O products in which the mobile phase is an aqueous solution of an organic acid.

This invention relates to novel biological stain compositions that areadapted for human in vivo topical application.

In particular the invention contemplates novel Toluidine Blue O ("TBO")dye products, products which contain TBO and specific TBO derivatives,in specific proportions.

According to another aspect, the invention pertains to new methods ofmanufacturing TBO compositions, including these novel TBO products.

In yet another aspect, the invention concerns in vivo methods of usingsuch novel TBO compositions to identify suspect dysplastic, i.e.,abnormal, tissue.

In still another and further aspect, the invention pertains tocompositions, in vivo diagnostic methods of use thereof and processesfor manufacture thereof, which are specially adapted for detectingsuspect dysplastic oral tissue, especially cancerous and precanceroustissue.

The various embodiments of the invention and the practice thereof willbe apparent to those skilled in the art, from the following detaileddescription thereof, taken in conjunction with the claims and inconjunction with the drawings, in which:

BACKGROUND OF THE INVENTION

Most oral lesions result from trauma. However, other oral lesions aredysplastic tumors, some of which may be benign, but some of which may beeither cancerous or precancerous. In addition, many dysplastic lesionsare small and easily missed on routine visual examination by dentalclinicians.

An in vivo diagnostic test is known which identifies and delineatessuspect dysplastic oral tissue. This screening test is generallydescribed in the U.S. Pat. No. 4,321,251 to Mashberg and in the U.S.Pat. No. 5,372,801 to Tucci et al. More recently kits have beendeveloped which make it possible for clinicians to quickly and easilyadminister the test, as part of other routine dental procedures, andthus identify and/or delineate suspect sites at a time when the patientsare symptomless or while the dysplastic lesions are so small that theymight be missed during normal visual examination. Once a suspectdysplastic lesion is identified by the Mashberg protocol, a regularbiopsy sample can be taken and subjected to histological examination, todetermine whether the lesion is malignant or precancerous. Kits forperforming this test, containing premixed dye and rinse solutions in theproper quantities and concentrations, are licensed by Zila, Inc. and areavailable commercially in Canada from Germiphene, Inc. under thetrademark ORASCAN™ and in The United Kingdom and Australia fromStafford-Miller Ltd. under the trademark "ORASCREEN®".

THE PRIOR ART

It has now been discovered that the organic dye content of the prior artTBO products which were typically commercially available, depending onthe vendor, was relatively low. Typically, the combined areas of the 254nm HPLC peaks (see HPLC procedure of Example 3),which represent theconformational isomers of TBO in these prior art products was only about≅2%-75% of the combined areas of the 254 nm HPLC peaks which representall of the TBO and TBO-related components, i.e., the two conformationalisomers of TBO plus up to six TBO-related components.

Referring to FIG. 1, HPLC peaks 7 and 8 represent the two conformationalisomers of TBO (shown here as the chloride salt): ##STR1## HPLC peaks 5and 6 have been identified as the N-demethylated derivatives of the twoconformational isomers of TBO: ##STR2## HPLC peaks 2 and 3 have beenidentifed as the N,N-demethylated derivatives of the two conformationalisomers of TBO: ##STR3## The exact structures of the compoundsrepresented by HPLC peaks 1 and 4 have not been positively determined.

At any rate, in the TBO compositions of the prior art, a typical one ofwhich is represented in FIG. 1, the compounds represented by peaks 1-4were present in relatively higher amounts than those of the presentinvention (See FIG. 2) and the compounds represented by peaks 5-8 werepresent in relatively lower amounts than in the TBO products of thepresent invention. The two N-demethylated derivatives of theconformational isomers of TBO, represented by peaks 5 and 6, of theprior art (FIG. 1) were present in the relatively higher amounts,typically upwards of 20% of the organic dye content, than those whichare present in the TBO products of my invention.

The classic synthesis of TBO is exemplified in the U.S. Pat. No.418,055, issued Nov. 30, 1989, to Dandliker et al. This synthesis is aseries of three oxidation steps: (1) oxidation ofN,N-dimethyl-p-phenylenediamine, e.g., with potassium dichromate, toform 2-amino-5-dimethylaminophenyl thiosulfonic acid; (2) condensationof the thiosulfonic acid with o-toluidine, to form the correspondingindamine-thiosulfonic acid; and (3) ring closure of theindamine-thiosulfonic acid, e.g., in the presence of zinc chloride atboiling temperature for about 30 minutes, to form TBO. The reactionmixture is then cooled and the TBO product of the ring-closure reactionis complexed and salted out, e.g., by treatment with sodium chloride andzinc chloride, to precipitate the TBO complex, e.g., as an TBO/ZnCl₂complex. Purification may be accomplished by repeated re-solution andre-precipitation, e.g., by re-solution in hot aqueous zinc chloridesolution and re-precipitation with sodium chloride/zinc chloride.

As far as known, prior workers, who instigated the use of TBO for invivo identification of dysplasia, used the above-described prior art TBOproducts, i.e., compositions in which the conformational isomers of TBOplus the N-demethylation and N,N-demethylation derivatives were lessthan 80% of the dye composition and in which the two N-demethylationderivatives of the conformational isomers, formed greater than about 20%% of the dye composition. According to my information, prior workerswere unaware of the exact composition of their "TBO" products andmanufacturers of prior art TBO products were unable to reproduciblyprepare them. In fact, the prevalent literature description of thequality of TBO is "toluidine blue of good color value". The BiologicalStain Commission specifies an analytical titration procedure fordetermining only the "organic dye content" of the material. The priorart use of such loosely defined "TBO" resulted in anomalous clinicalobservations and serious problems in obtaining necessary regulatoryclearances to manufacture and market such products for use in humandiagnostic procedures.

BRIEF DESCRIPTION OF THE INVENTION

Briefly, the new compositions of matter embodying the invention are aTBO product, in which the conformational isomers of TBO and theN-demethylation derivatives of these conformational isomers comprise andare present in a ratio of the TBO isomers to their N-demethylationderivatives such that the ratio of the combined areas of the 254 nm HPLCpeaks representing the TBO isomers (determined in accordance with theHPLC procedure of Example 3) to the combined areas of the peaksrepresenting their N-demethylation derivatives is at least about 6:1.Thus, as depicted in FIG. 2, the combined area of the 254 nm HPLC peaks,representing the TBO conformational isomers (peaks 7 and 8), is at leastabout six times the combined area of the 254 nm HPLC peaks, representingtheir respective N-demethylation derivatives (peaks 5 and 6). In thepreferred embodiment of the invention, the components represented bypeaks 5, 6, 7 and 8 equals at least about 95% of the organic dye contentof the product. In the most particularly preferred embodiment, the peak8 (254 nm) area represents at least 58% of the organic dye content ofthe product.

The invention also contemplates a method for human in vivo detection ofdysplastic tissue, which includes the step of applying to human tissuethe above-described new TBO products.

Still another embodiment of the invention is a process for reproduciblymanufacturing TBO compositions, including the above-described novel TBOproducts, in which the complexing agent is added to the reaction mixturebefore the ring closure (third) step of the Dandliker synthesis,preferably before the first oxidation step (11, of FIG. 3) of theprocess.

The prior art Dandliker process included the steps of

oxidizing N,N-dimethyl-ρ-phenylene diamine ions in a first reactionmixture to form a first intermediate, 2-amino-5-dimethylaminophenylthiosulfonic acid,

oxidizing the first intermediate and condensation of the oxidizate witho-toluidine, in a second reaction mixture, to form a secondintermediate, indamine thiosulfonic acid,

oxidizing the second intermediate in a third reaction mixture to closethe indamine ring, forming a TBO reaction product, dissolved in thethird reaction mixture,

introducing a complexing reagent into said third reaction mixture, toform a TBO-complex product, and

separating said TBO-complex from said third reaction mixture.

My improvement on this prior art Dandliker process comprises the step ofadding the complexing reagent at a stage earlier than the formation ofthe third reaction mixture, preferably before the formation of thesecond reaction mixture.

According to a still further and presently preferred embodiment,specially adapted to manufacture the novel TBO product compositions ofthe invention, the temperature of the reaction mixtures during theoxidation steps is maintained at not greater than about 10° centigrade.

In yet another further and presently preferred embodiment, speciallyadapted to improve the quality of the TBO product, the pH of thereaction mixture during the oxidation steps is maintained in the rangeof about 2.8-3.8 (preferably 3.3) in the first reaction mixture, about3.1-4.1 (preferably 3.6) in the second reaction mixture and about 3.0 inthe third reaction mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a 254 nm HPLC chart, depicting the peaks which are typicallycharacteristic of TBO product compositions which were previously knownand commercially available;

FIG. 2 is a 254 nm HPLC chart, depicting the peaks which arecharacteristic of typical TBO product compositions of the presentinvention; and

FIG. 3 is a process flow diagram, depicting the process which I havediscovered, for manufacturing TBO products, including the novel TBOproduct compositions of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As depicted in FIG. 1, typical previously commercially available TBOexhibited two 254 nm HPLC peaks (peaks 7 and 8), which represent theconformational isomers of TBO, and two 254 nm HPLC peaks (peaks 5 and6), which have been discovered by me to be the demethylation derivativesof the conformational isomers. The relative quantities of theconformational isomers of TBO to their N-demethylated derivatives inthis typical prior art product was such that the peak area ratio of theconformational isomers to the N-demethylated derivatives is less than4:1. A higher ratio, approaching or exceeding 6:1, accidentally existedin isolated prior art TBO products, but the relative amounts of suchproducts were not known or considered important. In any event, TBOproducts with such higher ratios, could not be reproducibly prepared byprior art manufacturing procedures.

According to regulatory requirements based on recent clinical testing,TBO which is to be used in human diagnostic procedures (in generalaccordance with the Mashberg protocol) for detecting dysplastic tissue,must have an HPLC peak area ratio at 254 nm ratio of the conformationalTBO isomers to the N-demethylation derivatives of at least about 6:1,i.e., the area of the combined 254 nm HPLC peaks 7 and 8, must be atleast about six times greater than the area of combined 254 nm HPLCpeaks 5 and 6.

It would be highly desirable to provide TBO product compositions whichmeet the requirements for human diagnostic testing procedures, in whichthe ratio of areas of the 254 nm HPLC peaks of the conformationalisomers to the areas of their demethylation derivatives is at least 6:1.Further, it would be highly desirable to provide manufacturing processesfor reliably and reproducibly preparing such TBO products, having thisspecific ratio of TBO isomers to TBO demethylation derivatives, and forreliably and reproducibly producing other TBO products with increasedyield and overall purity.

As will be apparent from FIG. 2, which depicts a 254 nm HPLC analysis oftypical compositions of the present invention, the peak area ratio ofTBO conformational isomers to N-demethylation derivatives is greaterthan about 6:1, namely, 6.68:1, as demonstrated by the fact that thecombined areas of 254 nm. HPLC peaks 7 and 8 is 6.68 times greater thanthe combined areas of peaks 5 and 6. The overall purity, determined byHPLC and ignition tests, and defined as [one hundred minus thepercentage of ignition residue] multiplied by the HPLC purity (i.e., thesum of the areas of peaks 5, 6, 7 & 8 divided by the total peak areas)of the TBO products of the invention, is at least greater than 75%,compared to 2-10% for most of the prior art compositions. In isolatedincidents, a comparable purity may have been obtained in prior artproducts, but not reproducibly.

According to one aspect of my invention, the ratio of the areas of thepeaks (peaks 3, 6 & 8) with the ring methyl group in the 2-position(e.g., see Formula I) to the areas of the peaks (peaks 2, 5 & 7 )withthe ring methyl group in the 4-position (e.g., see Formula II) is˜2.5:1. By contrast, according to my knowledge, this ratio in the priorart products was no greater than 1.5:1.

This combination of high ratio of the areas of peaks 7+8: peaks 5+6 andthe high ratio of the areas of peaks 3+6+8: peaks 2+5+7 has not to myknowledge been exhibited by any prior art product. Since peak 8 is theprimary TBO peak, the structure of which is most widely accepted as TBO,then peaks 3, 6 and 8 are preferred to peaks 2, 5 and 7. Peaks 7 and 8are, of course, preferred over peaks 5 and 6, which are, in turn,preferred over peaks 2 and 3. Thus, in the most preferred embodiment ofmy invention, the products satisfy the combination of these two ratiocriteria.

FIG. 3 is a process flow diagram which depicts a process for preparingTBO products, which meet the regulatory requirements for clinical use ingeneral accordance with the Mashberg protocol.

The starting material 10 for the synthesis of FIG. 3 iscommercially-available, high-purity N,N-dimethyl,ρ-phenylene diamine:##STR4##

Formation of First Reaction Mixture

An aqueous solution of the starting material 10 is oxidized 11,preferably at less than 10° C., especially at less than about 5° C., byreaction with a suitable oxidizing agent 12, e.g., potassium dichromate12, in the presence of acid, aluminum sulfate and a reagent, 13 (whichis believed to complex the intermediate(s) and is used in a later stageof the process to complex the TBO composition components), e.g., zincchloride. Then, a source of thiosulfate ions 14, e.g., sodiumthiosulfate, is added to form a first reaction mixture 15 containing thefirst intermediate, 2-Amino-5-dimethylaminophenyl thiosulfonic acid:##STR5##

Formation of Second Reaction Mixture

The first reaction mixture 15 is then further reacted, preferrably at atemperature of not greater than about 10° C., with additional oxidizingagent 16, e.g., potassium dichromate, and o-toluidine hydrochloride 17,in a condensation step 18 to form the second intermediate, acondensation product, indamine thiosulfonic acid ##STR6## in the secondreaction mixture 19.

Formation of Third Reaction Mixture

The second reaction mixture 19 is then further oxidized 21, preferablyby addition of a suitable oxidizing agent 22, e.g., potassiumdichromate, at a temperature of not greater than about 10° C. This isfollowed by the addition of copper sulfate, zinc chloride complexingagent, acid and heating to 100° C. to effect closure of the indaminering, forming TBO in a third reaction mixture 24. At this point the TBOis separated from the third reaction mixture and purified.

Separation/Purification of TBO

For example, in the presently preferred embodiment of the process of thepresent invention, the TBO is precipitated from the third reactionmixture by complexation of 24 with a suitable complexing agent 25, e.g.,zinc chloride, to form the complex TBO-zinc chloride double salt. Theprecipitate is filtered 26 from the liquid phase and washed with sodiumchloride solution 27. The washed filter cake is then redissolved 28 in acritical¹ volume of water 29 to form a TBO solution 30, which is thenfiltered 31 to remove undissolved solids 32a, which are discarded. ZincChloride, followed by a critical² volume/concentration of sodiumchloride 33 is then added to the filtrate 32 to again precipitate theTBO-zinc

As indicated by the dashed line 35, the TBO filter cake 34 can beredissolved, filtered, re-precipitated and reisolated multiple times toachieve the desired degree of purity and yield of TBO. The finalpurified filter cake complex product 34 is then dried 35, e.g., inconventional convection oven and/or vacuum oven and the dried filtercake 36 is ground and blended 37 to yield the final TBO product 38. Thefinal TBO product contains both the zinc chloride double-salt of TBO(Formula X) and the chloride salt of TBO (Formulas I & II).

Introducing the complexing reagent before the formation of the thirdreaction mixture, i.e., prior to the oxidation of the indaminethiosulfonic acid and complexing the resultant TBO reaction product toform the TBO-complex, produces a TBO-complex product having an improvedratio of TBO conformational isomers to the N-demethylation productsthereof. If the complexing reagent is introduced before the formation ofthe third reaction mixture, a ratio of at least 6:1 can be obtained. Ofcourse, as will be recognized by those skilled in the art, obtainingthese improved ratios of isomers to demethylation derivatives will alsodepend somewhat on observing other processing parameter precautions, aswill be discussed below in connection with the disclosure of thepreferred embodiments of the process invention, which are desirable toimprove the yield and purity of the TBO-complex product. However, evenif such other yield and purity-increasing precautions are observed, thedesired improved ratios of isomers to N-demethylation products will notbe obtained and the desired improved ratio of peaks with the methylgroup in the -2 vs. -4 position will not be obtained, unless thecomplexing reagent is added at least before the formation of the thirdreaction mixture, i.e. before oxidizing the indamine sulfonic acid andcomplexing the resultant TBO product.

At present, I believe that the earlier addition of the complexingreagent, i.e., before the formation of the third reaction mixtureimproves the isomer:N-demethylation derivative ratio of the finalproduct, because of the early formation of a complex of the startingmaterial and/or the thiosulfonic acid and/or the indamine-thiosulfonicacid, which apparently offers steric hinderance to demethylation. Inother words, the complex, because of its increased size and structure,offers steric bulk (and possibly electronic effects) which protect theN-methyl groups from oxidative demethylation. Because all three reactionsteps involve oxidation and possible demethylation, the earliestformation possible of this complex is advantageous, which is why Irecommend that the complexing agent be present as early as possible.

WORKING EXAMPLES

The following examples are presented to illustrate the practice of theinvention in such terms as to enable those skilled in the art to makeand use the novel TBO compositions, to practice the novel diagnosticmethods using such TBO compositions and to practice the novel processfor preparing TBO compositions, which together form the variousembodiments of the invention, and to indicate to those skilled in theart the presently known best modes for practicing the variousembodiments of the invention. These examples are presented asillustrative only and not as indicating limits on the scope of theinvention, which is defined only by the appended claims.

Example 1 Manufacturing Process

This example illustrates, in the detail required to satisfy regulatoryrequired GMP conditions, the exact procedures for carrying out thecommercial scale manufacture of a batch of TBO dye product, according tothe process which embodies the presently known best mode of theinvention.

Preparation of Raw Materials Solutions

    ______________________________________                                        Equipment/supplies:                                                           ______________________________________                                        A.      Ohaus IP15KS Balance                                                  B.      AnD HV150KAI Balance                                                  C.      Fairbanks H90-5150 Balance                                            D.      OHAUS WB25/I-20W Balance                                              E.      Cole Parmer (51201-30) and Thermolyne                                         (S25535) Stirrers                                                     F.      Sampling devices, such as steel scoops, drum                                  samplers, etc.                                                        G.      Erlyenmeyer flasks, beakers, carboys and other                                appropriate glassware.                                                H.      Production Solution Labels.                                           ______________________________________                                    

Safety:

Protective equipment, such as gloves, safety glasses, lab coats, andrespirators should be worn when handling chemicals according to MSDSguidelines.

Raw Material Solutions Preparation Procedure:

To Hydrochloric Acid, 1364.2 g (±5.5 g) add 1364.2 g (±5.5 g) of USPPurified water. Stir until the solution is clear.

To Aluminum Sulfate Hexadecahydrate, 1779.1 g (±7.0 g) add 2548.9 g(±10.0 g) of USP Purified water. Stir until the solution is clear.

To Zinc Chloride, 7384.6 g (±30.0 g), add 2786.7 g (±11.0 g) of USPPurified water. Stir until the solution is clear.

To Potassium Dichromate, 2101.9 g (±8.0 g), add 25203.8 g (±100 g) ofUSP Purified water. Stir until the solution is clear.

To Sodium Thiosulfate Pentahydrate, 1526.6 g (±6.0 g), add 2043.6 g(±8.0 g) of USP Purified water. Stir until the solution is clear.

To Copper Sulfate Pentahydrate, 509.7 g (±2.0 g), add 1613.1 g (±6.0 g)of USP Purified water. Stir until the solution is clear.

To Sulfuric Acid, 600.0 g (±2.0 g), add 600.0 g (±2.0 g) of USP Purifiedwater. Stir until the solution is clear.

To Sodium Chloride, 70.4 kg (±250 g), add 234.4 kg (±850 g) of USPPurified water. Stir until the solution is clear.

Safety

Protective equipment, such as gloves, safety glasses, lab coats, andrespirators should be worn when handling chemicals according to MSDSguidelines.

Synthesis

Synthesis Equipment and Supplies:

LFE Control Panel (3000)

20 gallon Jacketed Glass Lined Purification Tanks with lid (E71224)

Two 100 gallon Jacketed Glass Lined Purification Tank with lids (P1,PT-001)(P2, L-13621)

FTS Recirculating Cooler (RC96C032) and 500 gallon Cold Storage Tank(500 CST)

Three Caframo Mixers (BDC-1850) (R1, 18500961)(P1, 18501148) (P2,18501173) with shaft and impeller

Lightning Mixer (L1U08) (201550)

Three Heat Exchangers (Gardner Machinery) (R1, 01960763) (P1, 01960764)(P2, 08950727)

Three 12 KW Jacket Fluid recirculators (Watlow, BLC726C3S 20)

Three Recirculation Pumps (Sta-Rite, JBHD-62S, C48J2EC15)

Masterflex Digital Peristaltic Pump (A94002806)

Masterflex Peristaltic Pump (L95003320)

Cole Parmer Peristaltic Pump (B96002074)

Neutsche Filtration unit (70-2038, 43421-1)

Two Buchner Filtration Units (Z11,624-6, Z10,441-8)

Siemens Vacuum Pump (F2BV2)

60 Gallon Glass Lined Collection Tank with lid (86854, E164-1186)

Air Compressor (DF412-2) (9502312538)

Flow Controller (3-5500) (69705069190)

Six Batch Controllers (3-5600) (#1,69705069191, #2, 69705069199, #3,69705069194, #4, 69705058829, #5, 69705058805, #6, 69705069195)

Six Flow Sensors (#1, 69704295165, #2, 69704024995, #3, 69704024994, #4,69704025027, #5, 69612178606, #6, 69703120990)

Four Diaphragm Pumps (M1)

Four Surge Suppressers (A301H) (#2, 15557, #3, 15561, #4, 15558, #5,15559)

Four Air Regulators (CFR10)

Four Solenoid Valves (used with air regulators)

Four Low Flow Sensors (FS-500)

Three Solenoid Valves (EASM5V16W20)

Air Filter / Regulator (T1R)PTFE / F06R113AC

Filter media, Polypropylene (7211-1)

Filter media, Whatman Grade 52

PharMed tubing (-18, -82, -90)

pH Meter; Hanna 9321 (1303675) & Orion 620 (001911)

Spectrophotometer 20 (3MU7202070)

Fisher Scientific Vacuum Oven (9502-033)

VWR 1370 FM forced air oven (1370 FM)

Dust/Mist Respirator

Thomas Wiley Laboratory Mill (3375-E10)

Patterson-Kelley Blender (Blendmaster, C416578)

OHAUS TS4KD Balance

OHAUS IP15KS Balance

Mettler AG 104 Balance

AnD HV150KA1 Balance

Fairbanks H90-5150 Balance

OHAUS AS123 Printer

OHAUS AS142 Printer

AD-8121 Multifunction Printer

Citizen iDP 3540 Dot Matrix Printer

Hewlett Packard HPLC (1050)

Ultrasonic Cleaner (8892-DTH, QCC9601 005C)

Type K Thermocouple Temperature Recorder (KTx, 6292753, 6355146)

Erlenmeyer Flasks (8 L, 6 L, 4 L, 1 L)

Beakers (8 L, 6 L, 500 mL, 250 mL)

Carboys (4 L, 10 L, 50 L)

HDPE Drums (55 gallon, 100 gallon)

Volumetric Flasks (100 mL)

Plastic Funnel

Pastuer Pipettes & Bulbs and Volumetric Pipettes (10 mL, 5 mL) & Bulb

Bellows (25 mL, 50 mL)

Weigh Paper

Spatulas

Packaging Material (containers, lids, labels)

Raw Material Solutions

SYNTHESIS: Step 1 Synthesis of 2-amino-5-dimethylaminophenylthiosulfonic acid

Check the integrity of the USP water system. To the reactor add theweighed USP Grade Purified Water (28,000 g ±100.0 g) and stir at 190±10RPM. Record the conductivity of the USP water at the time the water wasdispensed.

Add N,N-dimethyl-1,4-phenylenediamine (5.128 mol, 720.0 g±3.0 g). Thematerial should be added as a powder (no lumps). Stir 10 minutes (±5minutes).

Add hydrochloric acid (6 N, 1136.9 g ±5.0 g). Stir 15 minutes (±5minutes).

Ensure the pH meter is calibrated according to SOP #LM-007. Take areaction mixture sample of approximately 10 mL using a plastic samplingdevice. Mark the sample lot #.IPS1a. Check the pH and record. The pHmust be 2.8-3.8 @25° C.±5° C.

Add aluminum sulfate hexadecahydrate solution (4328.0 g ±21.0 g). Stir10 minutes (±5 minutes) at 275±10 RPM.

Add zinc chloride solution (3641.5 g ±18.0 g). Cool to 4° C.±1° C.

Once the temperature (PV1) is 4° C.±1° C. add potassium dichromatesolution (6532.4 g ±32.0 g) over a 20 minute period (±5 minutes). Whenaddition is complete stir 20 minutes (±5 minutes) and then change theSet Point (SP1) to 25.0° C. from the Main Menu.

When the temperature has reached 20.0° C. ±3.0° C. add sodiumthiosulfate pentahydrate solution (3570.2 g ±18.0 g). Stir the solutionat 25° C. for 30 minutes (±5 minutes).

Change the Set Point to 60° C. When the temperature (PV1) reaches 60.0°C. ±3.0° C. allow the reaction mixture to stir 5 minutes (±3 minutes)and change the Set Point on the LFE controller to 10.0.

Once the temperature has reached 13.0° C. ±2.0° C. take a reactionmixture sample of approximately 10 mL using a plastic sampling device.Mark the sample lot #.IPS1b. Check the pH and record. The pH must be3.1-4.1@25° C. ±5° C.

SYNTHESIS: Step 2 Synthesis of Indamine Thiosulfonic Acid

Weigh out o-toluidine (604.4 g ±2.5 g) and cool to 18° C. ±3° C. in anice bath. Add hydrochloric acid (6 N, 1230.7 g ±5.0 g) to theo-toluidine slowly. Remove the o-toluidine hydrochloride from the icebath and allow the solution to cool to 38° C. ±3° C. Add the solution tothe reaction mixture and stir 5 minutes (±3 minutes).

Add potassium dichromate solution (6532.4 g ±32.0 g) over a 20 minuteperiod (±5 minutes). When addition is complete stir 10 minutes (±5minutes).

Change the controller Set Point (SP1) to 60.0. Once the reaction mixturetemperature reaches 60.0° C. ±3° C. allow the mixture to stir 25 minutes(±5 minutes). A precipitate will form consisting of a green indamine.

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS2. Record the solution color.

SYNTHESIS: Step 3 Synthesis of Toluidine Blue O and Toluidine Blue OZinc Chloride Double Salt

Set the LFE controller Set Point to 7.0. Once the reaction mixturetemperature reaches 10.0° C. ±3° C. add potassium dichromate solution(6532.4 g ±32.0 g) over a 20 minute period (±5 minutes). When additionis complete stir 20 minutes.

Add potassium dichromate solution (5225.9 g ±26.0 g) over a 20 minuteperiod (±5 minutes). When addition is complete stir 20 minutes (±5minutes).

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS3.

Add zinc chloride solution (3641.5 g ±18.0 g). Stir 20 minutes (±5minutes) at 350 ±10 RPM.

Add copper sulfate pentahydrate (2122.8 g ±10.0 g). Stir 15 minutes (±5minutes).

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS4.

Change the controller Set Point (SP1) to 100.0. Once the reactionmixture temperature reaches 67.0° C. ±3° C. begin to add sulfuric acidsolution to pH 2.9±0.3 by adding aliquots (500 mL, 250, 125 mL, etc.).Stir for 5 to 10 minutes after each addition and check pH.

Once the reaction mixture temperature reaches 100.0° C ±3° C. allow themixture to stir 35 ±5 minutes.

Change the controller Set Point (SP1) to 35.0. Once the reaction mixturetemperature reaches 70.0° C. ±3° C. take a reaction mixture sample ofapproximately 10 mL using a pipette. Mark the sample lot #.IPS5.

Change the controller Set Point (SP1) to 2.5. Cool to 2.5° C. in 4 hoursand Hold at 2.5° C. ±2.0° C. for 4 to 18 hours.

Take a reaction mixture sample of approximately 10 mL using a pipette.Mark the sample lot #.IPS6. Record the solution color. Check the pH andrecord. Filter the sample through 0.45 micron filter paper. Takeapproximately 100 milligrams of the precipitate and dissolve inapproximately 100 mL of HPLC water. Filter the solution through 0.45micron filter paper. Label the solution Lot #.IPS7 and analyze thesample by the RP-HPLC Toluidine Blue O Analysis Method. See Example 3.Record the results.

Purification: Step 1

Filter the reaction mixture through suitable filter media (Whatman Grade52).

When the reactor is empty weigh out 24.0 kg ±150.0 g of 30% NaClsolution and add 24.0 kg ±150.0 g of USP water (record conductivity ofthe dispensed water). Close the reactor bottom valve and add the 15%NaCl solution to the reactor. Stir the solution briefly. When thefiltration is complete add the NaCl solution to the filtration unit torinse the filter cake. Collect the filtrate into the same container andLabel lot#.HW1 (hazardous waste 1).

Process filtrate (lot#. HW1) according to waste disposal procedures.

Check the 100 gallon glass lined, jacketed purification tank #1condition and make certain the tank has been properly labeled as CLEANEDwith date and signature. Equip the tank with a HDPE lid, Caframostirrer, stir shaft, propeller and thermocouple probe inserted into aplastic thermocouple well. Check that the bottom valve is off and theoutlet is capped.

Label the Tank with Lot#.P1A (Purification 1A).

Weigh out 190.0 kg ±1.0 kg of USP water into a HDPE container (recordconductivity of the dispensed water) and transfer the water toPurification Tank 1. Stir the mixture at 350 RPM. Once the NaCl wash ofthe filter cake is complete add the filter cake to Purification Tank 1while stirring.

Stir the mixture 2 to 4 hours. Take a sample (through the bottom valve)of approximately 50 mL. Mark the sample lot#.IPS8. Record the solutioncolor.

Set the Purification Tank 1 LFE controller to 75.0 (SP1).

When the mixture temperature (PV1) reaches 75.0° C. ±3° C. change theSet Point on the controller to 40.0.

Allow the mixture to stir at 40° C. and 350 RPM for 12 to 36 hours.

Take a sample (through the bottom valve) of approximately 50 mL. Markthe sample lot#.IPS9. Record the solution color. Check the pH andrecord. Measure 1.0 mL of the sample with a 1.0 mL pipette and dilute to100 mL in a volumetric 100 mL flask. Label the sample lot#.IPS9A. Thentake 10.0 mL of this solution with a 10.0 mL pipette and dilute to 100mL in a volumetric 100 mL flask. Label the sample lot#.IPS9B. Measurethe absorbance of these samples using the spectronic 20+. Record theresults. The absorbance of sample 9B should be ≧0.220.

Purification: Step 2

Filter the mixture through filter media in the filtration unit. Collectthe filtrate into a Tared HDPE container with lid.

Check the 100 gallon glass lined, jacketed purification tank # 2condition and make certain the tank has been properly labeled as CLEANEDwith date and signature. Equip the tank with a HDPE lid, Caframostirrer, stir shaft, propeller and thermocouple probe inserted into aplastic thermocouple well. Check that the bottom valve is labeled asCLEANED, off (horizontal position) and the outlet is capped.

Label the Tank with Lot#.P2A (Purification 2A), date and signature.

When the filtration is complete weigh the container and solution.Subtract the tare weight. Record the solution weight. Calculate thesolution volume.

    (TBO soln wt. g)(100.0 mL TBO soln/100.42 g TBO soln) =ml of TBO soln

Label the filter cake lot#.HW2 (Hazardous Waste 2) and process accordingto waste disposal procedures.

Into a clean HDPE container weigh out a quantity of 30% NaCl solutionequal to the solution volume recorded above using the following formula:

    (mL of TBO soln) (116.91 g NaCl soln/100.0 mL NaCl soln)=g of NaCl soln

Sample ≈10 mL of the filtrate and check the pH. Label lot#.IPS10. The pHmust be 3.0-4.0. Transfer the filtrate (by weight) to Purification Tank2. Stir the solution at 350 RPM.

Add zinc chloride solution (1636.3 g ±6.5 g)

Transfer the NaCl solution (by weight) to Purification Tank 2.

Set the Purification Tank 2 LFE controller to 75.0 (SP1).

When the mixture temperature (PV1) reaches 75.0° C. ±3° C. change theSet Point on the controller to 5.0.

Cool to 5° C. in 6 hours and Hold at 5° C. ±4.0° C. for 4 to 24 hours.

Take a sample (through the bottom valve) of approximately 50 mL. Markthe sample lot#.IPS11.PT2.

PROCESSING

i. Filter

Filter the mixture through tared filtration media (Whatman Grade 52) inthe filtration unit

Weigh out 12 kg ±50 g of 30% sodium chloride solution and dilute with 12kg ±50 g of USP water (record conductivity of the dispensed water). Washthe filter cake with the 15% sodium chloride solution by adding thesolution directly to the buchner. When the filtration is completecarefully remove the filter paper containing the toluidine blue Oproduct.

Process Lot#.HW3 (Hazardous Waste 3) according to waste disposalprocedures.

ii. Dry

Place the TBO product in the oven and dry at 50.0° C. ±3.0° C. for 5 ±1hours. Label the oven lot#.PRE-DRY.

Remove the product from the forced air oven and place in the VacuumOven. Dry at 45.0° C. ±3.0° C. @ 28" Hg ±2" Hg for 10 ±2 hours. Labelthe oven lot#.DRY.

iii. Weigh

Remove the product and weigh the Toluidine Blue O and filter. Subtractthe filter weight and record the TBO weight.

Using a stainless steel spatula carefully remove the product from thefilter paper. Wear a Dust/Mist respirator. Weigh the Toluidine Blue.

iv. Grind

Transfer the product to the TOLUIDINE BLUE O FINISHING AREA. Check theThomas Wiley Laboratory Mill condition and make certain the mill hasbeen properly labeled as CLEANED with date and signature. Use the 0.5 mmscreen. Attach a clean container to the delivery chute. The chamber doormust be closed and latched.

Close the sliding shutter at the bottom of the hopper, remove the hopperlid and add the sample. Replace the hopper lid. Turn the mill ON andopen the sliding shutter slightly. Feed sample into the mill chamberslowly enough so that the mill does not slow down or become jammed.

Once the grinding is complete carefully remove the mason jar from thedelivery chute.

v. Blend

Check the Patterson-Kelly Lab Blender condition and make certain theblender has been properly labeled as CLEANED with date and signature.

Transfer the Toluidine Blue O product to the blender container and closethe lid. Set the timer to 15 minutes ±5 min.

vi. Test

Sample the product for testing. Analyze the sample by the RP-HPLCToluidine Blue O Analysis Method. Record the results.

Example 2 Clinical Testing Protocol

Preparation of Clinical Test Solutions

This example illustrates the use of the TBO product of Example 1A in theidentification of oral dysplasia.

The TBO product of Example 1, raspberry flavoring agent (IFF RaspberryIC563457), sodium acetate trihydrate buffering agent and H₂ O₂ (30% USP)preservative (See U.S. Pat. No. 5,372,801), are dissolved in purifiedwater (USP), glacial acetic acid and SD 18 ethyl alcohol, to produce aTBO test solution, having the composition indicated in Table A:

                  TABLE A                                                         ______________________________________                                        Component       Weight %                                                      ______________________________________                                        TBO Product     1.00                                                          Flavor          .20                                                           Buffering Agent 2.45                                                          Preservative    .41                                                           Acetic Acid     4.61                                                          Ethyl Alcohol   7.48                                                          Water           83.85                                                                         100.00                                                        ______________________________________                                    

Pre-rinse and post-rinse test solutions of 1 wt. % acetic acid inpurified water, sodium benzoate preservative and raspberry flavor areprepared.

Clinical Protocol

The patient is draped with a bib to protect clothing. Expectoration isexpected, so the patient is provided with a 10-oz. cup, which can bedisposed of in an infectious waste container or the contents of whichcan be poured directly into the center of a sink drain, to avoidstaining the sink. Environmental surfaces or objects which might bestained are draped or removed from the test area.

A visual oral cancer examination is conducted, without using anyinstruments which might cause nicks or cuts of soft tissues. Notationsare made of the pre-staining appearance of soft tissues and teeth.

The patient rinses the oral cavity with approximately 15 ml. of thepre-rinse solution for approximately 20 seconds and expectorates, toremove excess saliva and provide a consistent oral environment. Thisstep is then repeated with additional pre-rinse solution.

The patient then rinses and gargles with water for 20 seconds andexpectorates.

The patient then rinses and gargles with 30 ml. of the TBO test solutionfor one minute and expectorates.

The patient then rinses with 15 ml. of the post-rinse solution for 20seconds and expectorates. This step is then repeated.

The patient then rinses and gargles with water for 20 seconds andexpectorates. This step is then repeated.

Observations of the oral cavity are then made, using appropriatesoft-tissue examination techniques, including retraction, well-balancedlighting and magnification, if necessary. The location, size,morphology, color and surface characteristics of suspect lesions, thathave retained blue coloration are made and recorded.

In order to reduce false positives, the patient is brought back after10-14 days for a repeat of the above protocol. This period allows timefor healing of any ulcerative or traumatic lesion or irritating etiologyat the time of the first examination. A positive stain after the secondexamination of a suspect area detected in the first examination isconsidered an indication of cancerous or precancerous tissue and abiopsy is made to confirm this conclusion.

Early erythroplastic lesions stain blue, often in a stippled or patchypattern. However, it normal for the stain to be retained by theirregular papiliar crevices on the dorsum of the tongue, which is not apositive indication. Other areas which retain blue stain, but are notregarded as positive include dental plaque, gingival margins of eachtooth, diffuse stain of the soft palate because of dye transferred fromthe retained stain on the dorsum of the tongue, and ulcerative lesionswhich are easily distinguished. In all instances, where a lesion ishighly suspect, but does not stain positively with this test, it isnevertheless imperative that a biopsy be taken.

Example 3 HPLC Procedure

This example describes a procedure for analysis of TBO samples, for usein identifying, assaying and purity testing.

    ______________________________________                                        Equipment and Supplies                                                        ______________________________________                                        Acetonitrile, HPLC Grade                                                      Glacial Acetic Acid, reagent grade                                            Ammonium Acetate, reagent grade                                               Deionized Water, suitable for HPLC analysis                                   pH Meter, with standard pH 4.0 and 7.0 buffers                                Laboratory glassware, including volumetric flasks and                         pipettes                                                                      Ultrasonic Bath                                                               Analytical Balance                                                            Magnetic Stirrer                                                              Compressed Helium                                                             Filtration Apparatus with 0.45 micron nylon filters                           100 μL Syringe                                                             ______________________________________                                    

HPCL Chromatograph and Accessories, including

Hewlett Packard Series 1050 pump, or equivalent capable of isocratichigh-pressure flow

Hewlett Packard Series 1050 Diode Array Detector, or equivalentwavelength UV detector

Hewlett Packard Vectra Series 3 Disk Drive (computer controller) withUltra VGA 1280 Monitor and laser printer, or equvalent integratingrecorder

Prodigy, 5 μ, QDS (3) 100Å, 2.5 cm×4.6 mm, or equivalent HPLC column

Fixed-loop injector (10 or 20 AL)

Column Heater

Preparation of the Mobile Phase

Prepare one liter of 0.01 M ammonium acetate solution by transferring0.77 g of ammonium acetate to a 1000 mL volumetric flask. Add water, mixto dissolve and dilute to mark with water. Transfer the 0.010 M ammoniumacetate solution to an Earlenmeyer flask and stir with magnetic stirrer.Using pH meter previously calibrated with pH 4.0 and 7.0 buffers, adjustpH of solution to between 3.3 and 3.6 with acetic acid. Filter thesolution through a 0.45 μfilter. Filter acetonitrile through 0.45 μnylon filter, using Millipore filtration apparatus and add exactly 250mL to the stirred aqueous ammonium acetate solution. Place thisreservoir of the mobile phase in position for access by the HPLC pumpand purge with helium for 5-10 minutes.

Preparation of TBO Samples

Accurately weigh about 50 mg of TBO sample, transfer to a 100 mLvolumetric flask and dilute to the mark with water. Cap the flask,sonicate for 30 minutes and mix. This is a stock solution of about 0.5mg/mL.

Transfer 10.0 mL of the stock solution to a 100 mL volumetric flask,dilute to the mark with water and mix. This approximately 0.05 mg/mldiluted TBO working solution is labeled appropriately.

Chromatographic Conditions

Injection volume--10 or 20 μL

Flow rate--about 1.5 mL/minute

Column Temperature--40° C.

Detector wavelength--254 nm

Sensitivity and attentuation settings: appropriate for instrument used

Integration--Area Response

HPLC Analysis of Samples

Set up and allow HPLC to equilibrate with the mobile phase flow. Systemsuitability tests as per USP XXIII are used to verify the precision andaccuracy of the HPLC data obtained. For each assay, evaluate thefollowing parameters:

    ______________________________________                                        Precision:   A minimum of five injections of the                                           working sample are compared. The                                              Relative Standard Deviation (RSD) must                                        be equal to or less than 2.0%. Six                                            injections are made if the RSD is                                             greater than 2.0%, but less than 3.0%                                         for the combined areas of peaks 5-8.                             Resolution:  Calculate the baseline resolution of                                          peaks 7 and 8 (the major peaks) on one                                        chromatogram of the sample by the                                             following equation:                                                            ##STR8##                                                                     where                                                                         t.sub.1 = retention time, in mm, of peak 7                                    t.sub.2 = retention time, in mm, of peak 8                                    w.sub.1 = peak width, in mm, of peak 7                                        w.sub.2 = peak width, in mm, of peak 8                           The resolution between peaks 7 and 8 must be                                  greater than 1.5.                                                             Tailing:     Measure the peak symmetry to insure the                                       quantitation of area under the peak is                                        accurate. Calculate the tailing factor (T)                                    for peaks 7 and 8 on one chromatogram of                                      the sample; by the following equation:                                         T = W.sub.0.05 ÷ 2f                                                     where                                                                            W.sub.0.05 =                                                                        width of peak                                                                 determined at 5% from                                                         the baseline peak                                                             of the peak height                                                      f =   distance between peak                                                         maximum and peak                                                              front at W.sub.0.05                                      T should be less than a factor of 3.                                          ______________________________________                                    

Record the chromatograms and determine the area response of the mainpeaks (5, 6, 7 and 8), as well as all impurity peaks which are detected(all peaks other than solvent front peaks) appearing in thechromatogram.

Calculations and Other HPLC Determinations

Identity (TBO drug substance and drug product):

The chromatographic profile of the sample preparation should show thesame general profile (peak presence and peak intensities) as that of thechromatogram depicted in FIG. 2.

Related Substances (drug substance):

The quantity of each impurity peak (the known impurities designated aspeaks 1, 2, 3 and 4 plus any other impurity peak) is calculated as anarea percent versus the total area of all peaks in the chromatogram.

Assay of the TBO drug substance:

The percentage of each of the four main tbo peaks (peaks 5-8) isdetermined as for the impurities, i.e., ##EQU1##

Having described my invention in such terms as to enable those skilledin the art to understand and practice it and, having identified thepresently preferred best modes thereof, I claim:
 1. A composition ofmatter, comprising: (A) the conformational isomers of toluidine blue O,the compounds having the structures ##STR9## and ##STR10## and (B) theN-demethylation derivative of said isomers, the compounds having thestructures ##STR11## and ##STR12## the ratio of the combined areas ofthe 254 nm HPLC peaks representing said isomers to the combined areas ofthe peaks representing said N-demethylation derivatives being at leastabout 6:1.
 2. A composition of matter, comprising:(A) a first group ofcomponents, comprising a conformational isomer of toluidine blue Ohaving the ring methyl group in the -2 position, the compound having thestructure ##STR13## an N-demethylation derivative thereof, the compoundhaving the structure ##STR14## an N,N-demethylation derivative thereof,the compound having the structure ##STR15## and (B) a second group ofcomponents, comprising a conformational isomer of toluidine blue Ohaving the ring methyl group in the -4 position, the compound having thestructure ##STR16## an N-demethylation derivative thereof, a compoundhaving the structure ##STR17## an N,N-demethylation derivative thereof,the compound having the structure ##STR18## the ratio of the combinedareas of the 254 nm HPLC peaks representing said first group to thecombined areas of the 254 nm HPLC peaks representing said second groupbeing at least about 2.5:1.
 3. A composition of matter, comprisingconformational isomers of toluidine blue O, namely the compounds havingthe structures ##STR19## and ##STR20## in which said isomer ofsubparagraph (a) comprises at least 58% of the total organic dye contentof said composition.
 4. In a method for identification of dysplastictissue, which method includes applying a biological stain to humantissue which selectively stains dysplastic tissue. the improvementcomprising the step of applying to human oral tissue the composition ofclaim 1 in a liquid carrier.
 5. In a process for manufacturing toluidineblue O, which process comprises the steps of(A) oxidizingN,N-dimethyl-ρ-phenylene diamine in a first reaction mixture, to form afirst intermediate, 2-amino-5-dimethylaminophenyl thiosulfonic acid, (B)oxidizing said first intermediate and condensing the oxidizate in asecond reaction mixture with o-toluidine, forming a second intermediate,indamine thio-sulfonic acid, (C) oxidizing said second intermediate toclose the indamine ring thereof, forming a TBO-containing reactionproduct dissolved in a third reaction mixture, (D) introducing acomplexing reagent into said third reaction mixture, to form aTBO-complex product dissolved in said third reaction mixture, (E)precipitating said TBO-complex product from said third reaction mixture,and (F) separating said TBO-complex product, containing theconformational isomers of TBO, the compounds having the structures##STR21## N-demethylation derivatives of said isomers, compounds havingthe structures ##STR22## N,N-demethylation derivative of said isomers,compounds having the structures ##STR23## from said third reactionmixture, the improved process comprising introducing said complexingreagent to a reaction mixture before the formation of said thirdreaction mixture, said complexing reagent being a compound that formswith said N,N-dimethyl-ρ-phenylenediamine, said first intermediate,and/or said second intermediate, a complex that provides sterichinderance to demethylation thereof.
 6. The process of claim 5 in whichthe temperature of the reaction mixtures during the oxidation steps ismaintained at not higher than about 10° C.
 7. The process of claim 5 inwhich the pH of the reaction mixtures is maintained in the range ofabout 2.8 -3.8 in the first reaction mixture, about 3.1-4.1 in thesecond reaction mixture and about 3.0 in the third reaction mixture. 8.A compound which a member selected from the group consisting ofN-demethylated derivatives of the conformational isomers of toluidineblue O, from the group consisting of the compounds having the structuralformulas ##STR24## and ##STR25##
 9. A compound which is a memberselected from the group consisting of N,N-demethylated derivatives ofthe conformational isomers of toluidine blue O, from the groupconsisting of the compounds having the structural formulas and##STR26##10.
 10. In an HPLC method for analysis of a toluidine blue Odye product, said method including forming a mobile phase,forming a TBOsample solution, equilibrating an HPLC column with the mobile phaseflow,and injecting the sample solution into the HPLC column,theimprovement for identifying sample dye components and for assaying anddetermining the purity of said sample, said improvement comprisingforming said mobile phase as a composition comprising a water-solublesalt of an organic acid.